PROJECT SUMMARY The E2F1 transcription factor is deregulated in most cancers through disruptions in the p16INK4A-cyclin D-RB tumor suppressor pathway but the role E2F1 plays in cancer is complex as it can either promote or inhibit tumor development depending on the context. E2F1 can also have opposing activities at the molecular level by activating or repressing transcription and at the cellular level by promoting proliferation or apoptosis. Moreover, E2F1 can directly stimulate DNA repair independent of its role in transcription. A long-term goal of these studies is to understand how the various functions of E2F1 are regulated in the cell and how each activity contributes to the development or suppression of cancer. E2F1 is phosphorylated, acetylated, and methylated in response to DNA damage but how these modifications alter the activities of E2F1 remains poorly understood. This proposal is based on the hypothesis that post-translational modifications on E2F1 are read by interacting proteins to regulate E2F1 localization and functions in DNA repair, transcription and tumorigenesis. The TopBP1 protein specifically binds to E2F1 when it is phosphorylated by the ATM/ATR kinases and this interaction results in the recruitment of E2F1 to sites of DNA damage. Aim 1 studies will use a novel knock-in mouse model (S29A) that blocks E2F1 phosphorylation to determine the function of E2F1 and its associated proteins in dynamically modifying chromatin structure at sites of DNA damage to facilitate repair. Preliminary data demonstrates that E2F1 acetylation creates a binding motif for the bromodomains of the CBP and p300 acetyltransferases. A novel knock-in mouse model that blocks E2F1 acetylation will be used to study how the E2F1-CBP/p300 interaction regulates histone acetylation and modifies chromatin structure at sites of DNA damage to facilitate repair. These knock-in mice will also be used to examine the role of E2F1 acetylation in regulating transcription and suppressing tumor development (Aim 2). The TDRD3 protein was identified as a potential reader of an E2F1 arginine methylation mark (R109me). The role of TDRD3 in regulating E2F1-dependent functions in DNA repair and transcription will be studied and, if warranted, a knock-in mouse model will be developed to establish the role of this modification in modulating tumorigenesis. Together, these studies will reveal how the E2F1 post-translational modification code is read by various chromatin-modifying activities to regulate both DNA repair and transcription. Determining how these E2F1 post-translational modifications impact the development of cancer will reveal new opportunities to modulate the activities of E2F1 to inhibit tumor growth and enhance the effectiveness of therapies.